Botanical Antioxidants

ABSTRACT

A botanical extract that exhibits antioxidant activity, wherein the botanical extract is at least an extract from the leaf of a plant from the genus Vaccinium.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Patent ApplicationNo. 62/728,113, filed 7 Sep. 2018, the disclosure of which isincorporated herein in its entirety by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention generally relates to inhibitors ofoxidation-induced DNA damage, and more particularly to botanicalinhibitors of oxidation-induced DNA damage, namely, cranberry (Vacciniummacrocarpon) leaves, and the use of such plant-based inhibitors as anantioxidant.

Oxygen is a highly reactive atom that is capable of becoming part ofpotentially damaging molecules called “free radicals”. Free radicals,commonly known as reactive oxygen species (‘ROS’), contain one or moreunpaired electrons in their outermost orbital. Common examples ofreactive oxygen species include peroxyl radical (ROO^(●)), superoxideanion (O₂ ^(●)), reactive hydroxyl (OH^(●)), and hydrogen peroxide(H₂O₂) radicals. These free radicals are generated spontaneously inliving organisms during metabolism. As free radicals are highlyunstable, they react with other molecules in their vicinity (e.g.,proteins, lipids, or DNA) to attain stability by taking electrons fromthose molecules, thereby causing damage to the cell and initiating achain reaction of free-radical generation.

An imbalance between the generation of free radicals and cellularantioxidant can lead to oxidative stress. Oxidative stress occurs whenan oxygen molecule splits into single atoms with unpaired electrons,which are called free radicals. Since electrons prefer to be in pairs,these free radicals scavenge the body to seek out other electrons inwhich to pair with, causing damage to cells, proteins, and DNA in doingso. The term oxidative stress is used to describe the condition ofoxidative damage resulting when the critical balance between freeradical generation and antioxidant defenses is unfavorable.

Oxidative stress, arising as a result of an imbalance between freeradical production and antioxidant defenses, is associated with damageto a wide range of molecular species including lipids, proteins, andnucleic acids. An excess of oxidative stress can lead to the oxidationof lipids and proteins, which is associated with changes in theirstructure and functions. Short-term oxidative stress may occur intissues injured by trauma, infection, heat injury, hypertoxia, toxins,and excessive exercise. These injured tissues produce increased radicalgenerating enzymes (e.g., xanthine oxidase, lipogenase, cyclooxygenase)activation of phagocytes, release of free iron, copper ions, or adisruption of the electron transport chains of oxidativephosphorylation, producing excess ROS. Oxidative stress has beenimplicated in the etiology of several degenerative diseases, such asstroke, Parkinson's disease, Alzheimer's disease, rheumatoid arthritis,diabetes mellitus, peptic ulcer, gene mutations and cancer, heart andblood disorders, and inflammatory diseases. Oxidative stress is nowthought to make a significant contribution to all inflammatory diseases(arthritis, vasculitis, glomerulonephritis, lupus erythematous, adultrespiratory diseases syndrome), ischemic diseases (heart diseases,stroke, intestinal ischema), hemochromatosis, acquired immunodeficiencysyndrome, emphysema, organ transplantation, gastric ulcers, hypertensionand preeclampsia, neurological disorder (Alzheimer's disease,Parkinson's disease, muscular dystrophy), alcoholism, smoking-relateddiseases, and many others.

Antioxidants are capable of stabilizing, or deactivating, free radicalsbefore they attack cells. Application of an external source ofantioxidants can assist in coping with oxidative stress. These includesynthetic antioxidants such as butylated hydroxytoluene and butylatedhydroxyanisole; however, these synthetic antioxidants have recently beenreported to be dangerous for human health. Thus, the search foreffective, nontoxic, natural compounds with antioxidative activity hasintensified in recent years.

Antioxidants are reducing agents, examples of which includenutrient-derived antioxidants such as ascorbic acid (Vitamin C),tocopherols and tocotrienols (Vitamin E), carotenoids, and polyphenols;antioxidant enzymes such as superoxide dismutase, glutathioneperoxidase, and glutathione reductase; metal binding proteins such asferritin, lactoferrin, albumin, and ceruloplasimin; and trace metals(e.g., zinc and molybdenum). These antioxidants can scavenge reactiveoxygen species and inhibit the chain reaction by donating an electron tothe free radical. The antioxidant defense system, supported by dietaryantioxidants, protects the body from free radicals. However, duringoxidative stress, antioxidants are insufficient to maintain homeostasis.In such instances, antioxidants can be given as supplements, theconsumption of which can significantly reduce the risk for freeradical-associated diseases.

Phytomedicine plays an important role in the management of most of thesediseases, with plants being a potential source of natural antioxidants.Studies have shown that the consumption of polyphenolic compounds foundin tea, fruits, and vegetables is associated with low risk of thesediseases. Consequently, there is a growing research interest in plantsthat contain antioxidants and health-promoting phytoconstituents aspotential therapeutic agents. Medicinal plants provide a safe,cost-effective, ecological alternative to chemical antioxidants, whichcan be toxic on prolonged exposure.

Superfoods, functional foods, food supplements, and nutraceuticals havebeen introduced to the food industry and have enriched the productsthereof, contributing to its further growth. Berry fruits constitute alarge group of functional foods or “superfoods”, whose consumptiondelivers several health benefits beyond basic nutrition due to theirhigh content of bioactive natural products.

Cranberry (Vaccinium macrocarpon) was introduced to European settlers byNative Americans, who used the berries for treating kidney stones andurinary tract health problems. Since then, cranberry has been used totreat a variety of ailments, including urinary tract infections, stomachailments, scurvy, vomiting, and weight loss by a large portion of theNorth American population. There are a number of cranberry fruitextracts on the market, and cranberry fruit juice is a common andpopular beverage alone or in combination with other juices. Further,there is excellent recognition by the public of the health benefits ofcranberry fruit-based products.

A strong body of scientific research documents the contribution of theconsumption of berries to the three targets of functional foods: (a)health maintenance; (b) reduced risk of obesity; and (c) reduced risk ofchronic diet-related diseases (e.g., cardiovascular disease, type 2diabetes, and metabolic syndrome). In addition to the fruits, the leavesof berry plants have been used in traditional remedies. Leaf extractshave often been used against several diseases, such as colds, urinarytract inflammation, diabetes, and ocular dysfunction by Native Americansand other populations.

Still, little is known about the composition of leaves of berry plantsand their beneficial properties. It is known that the main bioactivecompounds in berry leaves are similar to those found in their fruits(i.e., phenolic acids and esters, flavonols, anthocyanins, andprocyanidins). It is also known that the concentrations of thesecompounds can vary from family to family within the genera Vaccinium.

As part of a healthy lifestyle and a well-balanced, wholesome diet,antioxidant supplementation is recognized as an important means ofimproving free radical protection. As noted above, there is a need foreffective, nontoxic, natural compounds with antioxidant activity. Thepresent invention provides one such solution.

BRIEF SUMMARY OF THE INVENTION

Disclosed herein is a composition comprising the botanical extract ofthe leaf of Vaccinium macrocarpon, wherein the botanical extractinhibits γ-H2AX activity. The botanical extract can be present in thecomposition in an amount of about 1.0 μg/mL or greater. Preferably, thebotanical extract is present in an amount of about 1.0 μg/mL to about1000.0 μg/mL.

Also disclosed herein is a dietary supplement having antioxidantproperties. The supplement comprises a cranberry leaf extract in atherapeutically effective amount, wherein the cranberry leaf extractinhibits γ-H2AX activity. The cranberry leaf extract is present in thesupplement in an amount of about 25.0 μg/mL to about 500.0 μg/mL.

The present invention further provides a method of inhibitingoxidation-induced DNA damage in a subject by administering a compositioncomprising the botanical extract of the leaf of Vaccinium macrocarpon ata concentration of about 1.0 μg/mL to about 1000.0 μg/mL. Preferably,the botanical leaf extract is present in the composition in an amount ofabout 25.0 g/mL to about 500.0 μg/mL.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 provides the chemical structures of various procyanidin andflavonoid compounds identified in cranberry fruit extract (E1)(non-exhaustive).

FIG. 2 provides the chemical structures of various procyanidin andflavonoid compounds identified in cranberry leaf extract (E2)(non-exhaustive).

FIG. 3 is an LC/MS TIC chromatogram of cranberry fruit extract (E1).

FIG. 4 is an LC/MS TIC chromatogram of cranberry leaf extract (E2).

FIG. 5 is LC/PDA (wavelengths of 280 and 350 nm) chromatograms ofcranberry fruit extract (E1).

FIG. 6 is LC/PDA (wavelengths of 280 and 350 nm) chromatograms ofcranberry leaf extract (E2).

FIG. 7 is LC/MS TIC chromatograms comparison between cranberry fruitextract (E1) and cranberry leaf extract (E2).

FIG. 8 provides the chemical structures of five anthocyanins identifiedin cranberry fruit extract (E1) present in the extract in an amount of1.90 mg/g total anthocyanins.

FIG. 9 is an illustration of the calibration curves of anthocyanins incranberry fruit extract (E1).

FIG. 10 is a graph illustrating the efficacy of cranberry fruit extract(E1) and cranberry leaf extract (E2) in inhibiting DNA damage comparedto catechin and soliprin standards.

FIG. 11 is a graph illustrating cell viability of a cell culture whendosed with various doses of cranberry fruit extract (E1) and cranberryleaf extract (E2) versus catechin and soliprin standards.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is a botanical extract of the fruit and/or leaf of aplant comprising multiple procyanidins and bioflavonoids, wherein thefruit extract has been standardized to an anthocyanin content of about1.90 mg/g, based on total weight of cyanidin-3-galactoside,cyaniding-3-arabinoside, peonidin-3-galactoside, peonidin-3-arabinoside,and malvidin-3-galactoside in the fruit extract, and wherein thebotanical extract comprises at least an extract from the genusVaccinium.

The present invention is further based on the surprising discovery thatthe leaf of the cranberry plant (Vaccinium macrocarpon) is substantiallyhigher in certain flavonoids than the cranberry fruit. In particular,the extract from the leaves has a flavonoid content of at least 20 timesgreater than the flavonoid content of the fruit of the cranberry plant.In another embodiment, the extract from the leaves comprises aprocyanidin trimers and procyanidin tetramers content of at least 23times and 700 times greater than the procyanidin trimers and procyanidintetramers content, respectively, of the fruit of the cranberry plant.Accordingly, in one embodiment, the botanical extract is from at leastthe leaves of Vaccinium macrocarpon. Further, the botanical extract isfrom at least the leaves of Vaccinium macrocarpon and exhibitsantioxidant activity.

When the botanical extract is at least the leaf of the plant, thebotanical extract can be present in the composition in an amount ofabout 1.0 μg/mL or greater. For example, the leaf extract can be presentin the composition in an amount of about 1.0 μg/mL to about 1000.0μg/mL.

For the present application, the term “composition” refers to a productthat treats, improves, promotes, increases, manages, controls,maintains, optimizes, modifies, reduces, inhibits, or prevents aparticular condition associated with a natural state, biological processor disease or disorder. For example, a composition improves theinhibition of oxidation and/or reduces inflammation, and the like in asubject. The term composition includes, but is not limited to,pharmaceutical (i.e., drug), over-the counter (OTC), cosmetic, food,food ingredient or dietary supplement compositions that include aneffective amount of an extract, at least one component thereof, or amixture thereof. Exemplary compositions include cream, cosmetic lotion,pack or powder, or as an emulsion, lotion, liniment foam, tablets,plasters, granules, or ointment. Compositions can also includebeverages, for example, beverages infused with an effective amount of anextract, or a tea satchel containing an effective amount of an extract.Non-limiting examples of food compositions containing an effectiveamount of an extract include baked goods, protein powders, meatproducts, dairy products, and confectionary.

As used herein, the term “extract” or “botanical extract” refers to asolid, viscid, or liquid substance or preparation that includes one ormore active ingredients of a substance of at least the plant Vaccinium(e.g., Vaccinium macrocarpon and/or Vaccinium oxycoccos). Preferably,the active ingredient is derived from the extract of the leaf of theplant. The extract can be prepared using a solvent such as water, loweralcohols of 1 to 4 carbon atoms (e.g., methanol, ethanol, butanol,etc.), ethylene, acetone, hexane, ether, chloroform, ethylacetate,butylacetate, dichloromethane, N,N-dimethylformamide (‘DMF’),dimethylsulfoxide (‘DMSO’), 1,3-butylene glycol, propylene glycol, andcombinations thereof, but also a fraction of the crude extract in such asolvent. So long as it assures the extraction and preservation of theactive ingredient(s), any extraction method may be employed.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” of a pure compound, composition, extract, extractmixture, component of the extract, and/or active agent or ingredient, ora combination thereof refers to an amount effective at dosages and forperiods of time sufficient to achieve a desired result. For example, the“effective amount” or “therapeutically effective amount” refers to thatamount of a pure compound, composition, extract, botanical extract,extract mixture, botanical extract mixture, component of the extract,and/or active agent or ingredient, or a combination thereof of thisinvention which, when administered to a subject (e.g., mammal, such as ahuman), is sufficient to effect treatment, such as improving theinhibition of oxidation and/or reducing inflammation, and the like in asubject. The amount of a composition, extract, botanical extract,extract mixture, botanical extract mixture, component of the extract,and/or active agent or ingredient of this disclosure that constitutes an“effective amount” or “therapeutically effective treatment” will varydepending on the active agent or the compound, the condition beingtreated and its severity, the manner of administration, the duration oftreatment, or the age of the subject to be treated, but can bedetermined routinely by one of ordinary skill in the art having regardto his own knowledge and to this disclosure.

The term “pharmaceutically acceptable” means those drugs, medicaments,extracts or inert ingredients, which are suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,incompatibility, instability, irritation, and the like, commensuratewith a reasonable benefit/risk ratio.

The terms “administer,” “administered,” “administers” and“administering” are defined as providing a composition to a subject viaa route known in the art, including but not limited to intravenous,intraarterial, oral, parenteral, buccal, topical, transdermal, rectal,intramuscular, subcutaneous, intraosseous, transmucosal, orintraperitoneal routes of administration. In preferred embodiments, oralroutes of administering a composition are suitable.

As used herein, the term “subject” or “individual” includes mammals towhich a composition may be administered. Non-limiting examples ofmammals include humans, non-human primates, canines, felines, equines,bovines, rodents (including transgenic and non-transgenic mice) or thelike. In some embodiments, the subject is a non-human mammal, and insome embodiments, the subject is human.

As used herein, the term “carrier” refers to a composition that aids inmaintaining one or more plant extracts in a soluble and homogeneousstate in a form suitable for administration, which is nontoxic and whichdoes not interact with other components in a deleterious manner.

Unless indicated otherwise, all proportions and percentages recitedthroughout this disclosure are by weight.

The present invention provides plant-based inhibitor capable ofinhibiting DNA damage due to oxidative stress. More particularly, thepresent invention is directed towards a botanical extract of the leavesof the cranberry plant from the genus Vaccinium. Such botanical extractshave been found to be capable of inhibiting oxidative stress-induced DNAdamage by neutralizing free radicals, thereby terminating the chainreaction created by the free radicals. By terminating the chainreaction, damage due to the free radicals by their reaction withimportant macromolecules such as DNA, protein, lipids, or the cellmembrane is prevented or inhibited.

Useful botanical extracts capable of inhibiting DNA damage due tooxidative stress according to the present invention include botanicalextracts from the genus Vaccinium. More particularly, the botanicalextract can be obtained from a plant chosen from Vacciniumarctostaphylos, Vaccinium macrocarpon, Vaccinium oxycoccos, Vacciniummicrocarpum, Vaccinium microcarpum, Vaccinium erythrocarpum, Vacciniumarboretum, Vaccinium crassifolium, Vaccinium angustifolium, Vacciniumboreale, Vaccinium caesariense, Vaccinium caespitosum, Vacciniumcorymbosum, Vaccinium darrowii, Vaccinium deliciosum, Vacciniumelliotii, Vaccinium floribundum, Vaccinium hirsutum, Vacciniummembranaceum, Vaccinium myrsinites, Vaccinium myrtilloides, Vacciniummyvrtillus, Vaccinium ovalifolium, Vaccinium ovatum, Vacciniumpadifolium, Vaccinium pallidum, Vaccinium parvifolium, Vacciniumpraestans, Vaccinium reticulatum, Vaccinium scoparium, Vacciniumstamineum, Vaccinium tenellum, Vaccinium uliginosum. Vaccinium virgatum,and/or Vaccinium vitis-idaea. Preferably, the botanical extract is atleast from Vaccinium macrocarpon. Vaccinium oxycoccos, Vacciniumnmicrocarpum, and/or Vaccinium microcarpum. More preferably, thebotanical extract is at least from Vaccinium macrocarpon; even morepreferably a botanical extract from the leaf of Vaccinium macrocarpon.

Compositions capable of inhibiting DNA damage due to oxidative stressaccording to the present invention may include one or more compoundsthat may function as active ingredients and which are a component of thebotanical extract. For example, the compound can be a phytochemicalpresent in the plant from which the plant extract is obtained. Thecompound may be at least partially responsible for inhibiting DNA damagedue to oxidative stress. The compound can be any compound capable ofinhibiting DNA damage due to oxidative stress. In one embodiment, thecompound is chosen from the phytochemicals isoquercetin,quercetin-3-glycoside, kaempferol glycoside, and/or procyanidins (e.g.,A, B, trimer, tetramer).

Generally, one or more parts of a plant can be used to produce abotanical extract including, but not limited to, the root, the stem, theleaf, the flower, the fruit, the seed, and the testa of the seed. In thepresent invention, at least the leaf of the plant is used—alone or withother plant parts, particularly the fruit—to produce the plant extract.The fruit and leaf from the Vaccinium plant can be commercially obtainedfrom various sources. The extract of the fruit and leaf can be obtainedusing any suitable extraction technique.

In this regard, one or more parts of the plant, particularly the leaf ofthe Vaccinium plant, can be collected and milled. Thereafter, the milledmaterial can be extracted using a suitable solvent. The solvent can beremoved in a concentration step. For example, the extracted material canbe screened or filtered to create a supernatant and a cake. The cake canbe pressed to remove a substantial portion of the liquid, which can beadded to the supernatant. The cake can then be dehydrated and used as afiber source. The supernatant can be distilled to remove the solvent ora portion thereof, to form a plant extract liquid concentrate. Theremoved solvent can be recycled. The concentrate can be dried (e.g., byspray drying) to provide a dried plant extract. This dried plant extractcan be assayed and/or standardized as described herein. Preferably, thedried plant extract is derived from Vaccinium macrocarpon, particularlythe leaf of the plant Vaccinium macrocarpon.

Suitable solvents for the extraction process include water, alcohol, ormixtures thereof. Exemplary alcoholic solvents include, but are notlimited to, C₁-C₇ alcohols (e.g., methanol, ethanol, propanol,isopropanol, and butanol), hydro-alcohols or mixtures of alcohol andwater (e.g., hydroethanol), polyhydric alcohols (e.g., propylene glycoland butylene glycol), and fatty alcohols. Any of these alcoholicsolvents can be used in the form of a mixture. In one embodiment, theplant extract is extracted using ethanol, water, or a combinationthereof (e.g., a mixture of about 70% ethanol and about 30% water). Inanother embodiment, the plant extract is extracted using only water.

In one embodiment, the plant extract can be obtained using an organicsolvent extraction technique. In another embodiment, solvent sequentialfractionation can be used to obtain the plant extract. Totalhydro-ethanolic extraction techniques can also be used to obtain theplant extract. Generally, this is referred to as a lump-sum extraction.

Total ethanol extraction can also be used. This technique uses ethanolas the solvent. This extraction technique can generate a plant extracthaving fat soluble and/or lipophilic compounds in addition to watersoluble compounds.

Another example of an extraction technique that can be used to obtainthe plant extract is supercritical fluid extraction (‘SFE’). In thisextraction procedure, the material to be extracted may not be exposed toany organic solvents. Rather, carbon dioxide can be used as theextraction solvent—with or without a modifier—in super-criticalconditions (>31.3° C. and >73.8 bar). Those skilled in the art willappreciate that temperature and pressure conditions can be varied toobtain the best yield of extract. This technique can generate an extractof fat soluble and/or lipophilic compounds, similar to a total hexaneand ethyl acetate extraction technique.

The botanical extract generated in the process can include a broadvariety of phytochemicals present in the extracted material. Thephytochemicals can be fat soluble or water soluble. Following collectionof the extract solution, the solvent can be evaporated, resulting in theextract.

The botanical extract can be standardized to a specified amount of aparticular compound. For example, the botanical extract can bestandardized to a specified amount of an active ingredient orphytochemical present in the extract.

The amount of plant extract present in the oxidative stress-induced DNAdamage inhibiting composition can depend upon several factors, includingthe desired level of oxidative stress-induced DNA damage inhibition, theoxidative stress-induced DNA damage inhibiting level of a particularplant extract or component thereof, and other factors. Preferably, theplant extract is present in an amount of from about 0.005 wt % orgreater, for example, from about 0.005 wt % to about 99.00 wt %, basedon total weight of the composition.

The oxidative stress-induced DNA damage inhibiting composition caninclude one or more acceptable carriers. The carrier can aid in enablingincorporation of the plant extract into an oxidative stress-induced DNAdamage inhibiting composition having a suitable form for administrationto a subject. A wide number of acceptable carriers are known in the art,and the carrier can be any suitable carrier. The carrier is preferablesuitable for administration to animals, including humans, and can beable to act as a carrier without substantially affecting the desiredactivity of the plant extract and/or any active ingredient. The carriercan be chosen based upon the desired administration route and dosageform of the composition.

Suitable dosage forms include liquid and solid forms. In one embodiment,the composition is in the form of a gel, a syrup, a slurry, or asuspension. In another embodiment, the composition is in a liquid dosageform such as a drink shot or a liquid concentrate. In a furtherembodiment, the composition is present in a solid dosage form, such as atablet, a pill, a capsule, a dragée, or a powder. When in liquid orsolid dosage form, the composition can be in a food delivery formsuitable for incorporation into food for delivery. Examples of suitablecarriers for use in solid forms (particularly tablet and capsule forms)include, but are not limited to, organic and inorganic inert carriermaterials such as gelatin, starch, magnesium stearate, talc, gums,silicon dioxide, stearic acid, cellulose, and the like. The carrier canbe substantially inert.

As an example, silicified microcrystalline cellulose can be used as acarrier or binder. Silicified microcrystalline cellulose is a physicalmixture of microcrystalline cellulose and colloidal silicon dioxide. Onesuch suitable form of silicified microcrystalline cellulose is ProSolvSMCC® 90, available from Penwest Pharmaceutical Co., Patterson, N.J.Silicon dioxide, in addition to that provided by the silicifiedmicrocrystalline cellulose, may be added to the composition as aprocessing aid. For example, silicon dioxide can be included as aglidant to improve the flow of powder during compression in themanufacturing of solid dosage units, such as tablet.

In another embodiment, the carrier is at least a functional carrier suchas buckwheat or spelt. By the addition of functional carriers into thecomposition, additional benefits may be provided such as lower glycemicindex compared to standard carriers such as those mentioned above.Further, functional carriers can be allergan free (e.g., buckwheat), andby adding them into the production process, the botanical extracts ofthe invention may benefit from the flavonoids of these functionalcarriers, such as rutin and quercetin. Also, the high fiber content ofthese functional carriers may facilitate and regulate intestinaltransit. Finally, the added mineral benefit of selenium found in speltmay aid in metabolism.

The oxidative stress-induced DNA damage inhibiting composition caninclude other inert ingredients, such as lubricants and/or glidants.Lubricants aid in the handling of tablets during manufacturing, such asduring ejection from dies. Glidants improve powder flow during tabletcompression. Stearic acid is an example of an acceptablelubricant/glidant.

The oxidative stress-induced DNA damage inhibiting composition can bemade in solid dosage form, such as tablets and capsules. This formprovides a product that can be easily transported by an individual to aplace of eating, such as a restaurant, and taken prior to, during, orafter consumption of a foodstuff. The composition can be formulated intodosage units containing suitable amounts of the plant extract and/oractive ingredient that permit an individual to determine an appropriatenumber of units to take based upon appropriate parameters, such as bodyweight, foodstuff size, or carbohydrate (e.g., sugar) content.

In one embodiment, the botanical extract is present in the compositionin a therapeutically effective amount, such as an amount of about 1.0μg/mL or greater, preferably from about 1.0 μg/mL to about 1000.0 μg/mL,more preferably from about 15.0 μg/mL to about 1000.0 μg/mL, even morepreferably from about 25.0 μg/mL to about 500.0 μg/mL, and even morepreferably from about 40.0 μg/mL to about 150.0 μg/mL. The compositioncan be administered as a single dose, or in multiple doses. In oneexample, the compound is administered in up to three doses per day. Forexample, the compound may be administered prior to a meal, during ameal, or after a meal. In one embodiment, the composition is a dietarysupplement having antioxidant properties containing cranberry leafextract in a therapeutically effective amount.

The dosage can be chosen to provide a level of inhibitory effect in asingle unit that may be effective for some individuals and/or somefoodstuffs, while also allowing for relatively simple dosage increasesto provide other levels of inhibitory effects that can be effective forother individuals and/or other foodstuffs.

The inhibiting composition can be in a form adapted for oral ingestion.This form can be configured as a single dosage form intended to providea specified dose of the plant extract. For example, the single dosageform can be a powder, a pill, a tablet, a capsule, or a drink shot. Thesingle dosage form can include, for example, from about 1.0μg/mL toabout 1000.0 μg/mL of the plant extract. Other forms include food orbeverage compositions containing the plant extract.

EXAMPLES Examples—Materials and Chemical Profiling Example 1—Preparationof 70% Ethanol Extracts from Cranberry Fruit and Cranberry Leaf

Dried Cranberry fruit powder (Vaccinium macrocarpon) (60 g) was loadedinto three 100 ml stainless steel tubes and extracted twice using asolvent of 70% ethanol in DI water with a Thermo Scientific™ Dionex™ ASE350 Accelerated Solvent Extractor at a temperature of 80° C. andpressure of 1500 psi. The extract solution was automatically filteredand collected. The combined ethanol extract solution was evaporated witha rotary evaporator under vacuum to give a crude 70% ethanol fruitextract (E1).

Dried ground Cranberry leaf powder (Vaccinium macrocarpon) (140 g) wasloaded into seven 100 ml stainless steel tubes and extracted twice usinga solvent of 70% ethanol in DI water with a Thermo Scientific™ Dionex™ASE 350 Accelerated Solvent Extractor at a temperature of 80° C. andpressure of 1500 psi. The extract solution was automatically filteredand collected. The combined ethanol extract solution was evaporated witha rotary evaporator under vacuum to give a crude 70% ethanol leafextract (E2).

The extraction results are provided in the following Table 1—

TABLE 1 Extraction of Cranberry fruit and Cranberry leaf Plant ExtractPlant Extract Extraction Part ID Powder (g) Weight (g) Yield (wt %)Fruit E1 60 27.40 45.67% Leaf E2 140 23.75 16.96%

Example 2—Chemistry Profiling of Cranberry Fruit and Cranberry LeafExtracts

Flavonoid compounds present in the cranberry fruit extract E1 andcranberry leaf extract E2 were determined using ultra high pressureliquid chromatography (‘HPLC’) and mass spectrometry (ACQUITY® UPLCI-Class and XEVO® GS-XT-QT of system, both available from WaterCorporation, Milford, Mass. USA). The column used was an ACQUITY® UPLCHSS T3 2.1×100 mm, 1.8 μm, with a column temperature of 40° C. and asample temperature of 15° C. For the mobile phase, Solvent A was 10%acetonitrile (‘ACN’) in water (0.1% Formic Acid), and Solvent B was ACN.The acquisition range was 100-1500 Daltons (‘Da’), and the acquisitionmode was electrospray ionization (‘ESI’) (-). Table 2 below provides theHPLC conditions—

TABLE 2 HPLC condition for analyzing E1 and E2 extracts Run InjectionExtract Time (min) Volume (μL) Concentration E1 20.00 1.00 5 mg/mL E220.00 2.00 1 mg/mL

Peak identification was based on accurate mass only. Multiple isomersmay have been identified as the same compound due to the limitation ofthe database. For example, eight (8) procyanidin B1-B8 compounds havingthe same molecular weight of 578.528 were not differentiated in thisanalysis.

Procyanidins and flavonoid glycosides such as quercetin, isoquercetin,and myricetin 3-arabinofuranoside were detected and identified based onaccurate mass in E1 at relatively low content. Chemical structures ofcompounds detected in E1 (non-exhaustive) are illustrated in FIG. 1. Thefollowing table lists compounds identified in E1 based on accurate mass—

TABLE 3 Compounds Identified in E1 Observed Mass Neutral NeutralObserved error Observed Detector Compound Name Mass (Da) Mass (Da) m/z(ppm) RT (min) counts Vaccihein A 378.09508 378.0935 377.0862 −4.3 0.6522406 Procyanidin B 578.14243 578.1445 577.1373 3.6 0.66 138868-[5-(3,4-Dihydroxy-7- 510.13147 510.1291 509.1218 −4.7 0.68 21507hydroxy-4-oxo-2H-1- benzopyran-2-yl)-2- hydroxyphenyl]-2,3-dihydro-7-hydroxy-2-(4-hydroxyphenyl)- 4H-1-benzopyran-4-one Procyanidin trimer864.19016 864.1939 863.1867 4.4 0.72 19512 Monotropein 390.11621390.1165 389.1092 0.8 0.93 7503 Orcinol gentiobioside, 448.15808448.1574 447.1501 −1.6 3.20 22920 Anacardioside 2-O-Benzoylglucose;D-form 284.08960 284.0894 283.0822 −0.6 3.54 18514 Leptosin 462.11621462.1164 461.1091 0.4 3.59 51758 Leptosin 462.11621 462.1164 461.10910.4 3.63 38344 2-O-Benzoylglucose; D-form 284.08960 284.0893 283.0820−1.0 3.71 6747 Procyanidin trimer 864.19016 864.1872 863.1800 −3.4 3.795716 Dunalianoside B 450.11621 450.1150 449.1077 −2.7 3.95 7628Dunalianoside B 450.11621 450.1147 449.1074 −3.5 4.12 7014 Procyanidintrimer 864.19016 864.1862 863.1789 −4.6 4.15 45918 2-O-Benzoylglucose;D-form 284.08960 284.0891 283.0819 −1.6 4.17 6085 Procyanidin tetramer1152.25355 1152.2530 1151.2457 −0.5 4.37 5523 Procyanidin trimer864.19016 864.1866 863.1793 −4.1 4.98 5966 Myricetin 3-arabinofuranoside450.07983 450.0793 449.0721 −1.1 5.17 8296 Myricetin 3-arabinofuranoside450.07983 450.0795 449.0723 −0.6 5.51 16797 Myricetin3-arabiriofuranoside 450.07983 450.0803 449.0730 1.0 5.64 46613Vaccinoside 536.15299 536.1530 535.1457 0.0 5.78 28664 Vaccinoside536.15299 536.1533 535.1460 0.6 5.97 72372 Procyanidin A 576.12678576.1274 575.1201 1.1 6.13 119550 Monotropein; 6,7-Dihydro,10- 538.16864538.1692 537.1620 1.1 6.16 57726 O-(4-hydroxy-E-cinnamoyl) Monotropein;6,7-Dihydro,10- 538.16864 538.1699 537.1626 2.4 6.33 151522O-(4-hydroxy-E-cinnamoyl) Vaccinoside 536.15299 536.1536 535.1463 1.16.35 7992 Avicularin 434.08491 434.0858 433.0785 2.0 6.38 62923Vaccinoside 536.15299 536.1534 535.1461 0.7 6.46 5222 Avicularin434.08491 434.0860 433.0787 2.5 6.56 52683 Avicularin 434.08491 434.0859433.0787 2.4 6.79 130113 Myricetin 3′-methyl ether 332.05322 332.0536331.0463 1.1 9.83 10303 4-O-Acetyl-6-trans- 476.13186 476.1319 475.12470.1 12.14 12950 caffeoylarbutin

Abundant bioflavonoids were identified in E2, including avicularin,isoquercetin, kaempferol, glycosides, and others. Chemical structures ofcompounds detected in E2 (non-exhaustive) are illustrated in FIG. 2. Thefollowing table lists compounds identified in E2 based on accurate mass—

TABLE 4 Compounds Identified in E2 Observed Mass Neutral NeutralObserved error Observed Detector Compound Name Mass (Da) Mass (Da) m/z(ppm) RT (min) counts Procyanidin B 578.14243 578.1441 577.1368 2.8 0.6713416 Monotropein 390.11621 390.1155 389.1082 −1.8 0.72 31923Procyanidin trimer 864.19016 864.1872 863.1799 −3.4 0.75 9024Procyanidin tetramer 1152.25355 1152.2512 1151.2439 −2.0 0.75 33165Myricetin 3-arabinofuranoside 450.07983 450.0792 449.0720 −1.3 0.94 6589Monotropein 390.11621 390.1166 389.1093 0.9 0.94 43918 Procyanidintetramer 1152.25355 1152.2502 1151.2429 −2.9 2.36 28086 Procyanidin B578.14243 578.1411 577.1338 −2.3 3.19 10152 Orcinol gentiobioside,448.15808 448.1582 447.1510 0.3 3.19 480731 Anacardioside Procyanidintrimer 864.19016 864.1878 863.1806 −2.7 3.25 104158 Procyanidin tetramer1152.25355 1152.2502 1151.2429 −2.9 3.28 34709 Procyanidin A 576.12678576.1260 575.1188 −1.3 3.29 6558 Procyanidin B 578.14243 578.1418577.1345 −1.2 3.35 31488 Orcinol gentiobioside 448.15808 448.1581447.1508 0.1 3.41 55958 Procyanidin tetramer 1152.25355 1152.24931151.2420 −3.7 3.60 22964 Orcinol gentiobioside, 448.15808 448.1574447.1501 −1.5 3.63 9322 Anacardioside Procyanidin trimer 864.19016864.1872 863.1799 −3.5 3.80 53828 Dunalianoside B 450.11621 450.1157449.1084 −1.2 3.94 20828 Procyanidin trimer 864.19016 864.1883 863.1811−2.1 4.16 262966 Procyanidin tetramer 1152.25355 1152.2507 1151.2434−2.5 4.38 89683 Procyanidin A 576.12678 576.1261 575.1188 −1.2 4.3813405 Procyanidin trimer 864.19016 864.1870 863.1797 −3.6 4.54 9939Procyanidin trimer 864.19016 864.1885 863.1812 −2.0 4.98 98041Procyanidin A 576.12678 576.1262 575.1190 −0.9 4.99 9959 Procyanidin A576.12678 576.1257 575.1185 −1.8 5.10 22194 Procyanidin tetramer1152.25355 1152.2495 1151.2423 −3.5 5.14 21067 Procyanidin tetramer1152.25355 1152.2490 1151.2417 −4.0 5.26 14044 Procyanidin A 576.12678576.1264 575.1191 −0.7 5.26 7671 Procyanidin trimer 864.19016 864.1871863.1798 −3.6 5.34 9598 Procyanidin A 576.12678 576.1246 575.1173 −3.85.47 6853 Procyanidin tetramer 1152.25355 1152.2491 1151.2419 −3.8 5.4717471 Procyanidin trimer 864.19016 864.1873 863.1800 −3.3 5.53 11401Myricetin 3-arabinofuranoside 450.07983 450.0804 449.0732 1.4 5.63 22203Vaccinoside 536.15299 536.1531 535.1458 0.1 5.78 98913 Dunalianoside B450.11621 450.1164 449.1091 0.4 5.83 5653 Vaccinoside 536.15299 536.1531535.1459 0.3 5.97 153237 Procyanidin A 576.12678 576.1275 575.1202 1.36.12 398543 Procyanidin tetramer 1152.25355 1152.2502 1151.2429 −2.96.12 35819 Jeediflavanone 558.11621 558.1170 557.1097 1.4 6.12 5855Monotropein; 6,7-Dihydro,10-O-(4- 538.16864 538.1696 537.1623 1.7 6.15208791 hydroxy-E-cinnamoyl) Procyanidin trimer 864.19016 864.1890863.1817 −1.4 6.20 65398 Monotropein; 6,7-Dihydro,10-O-(4- 538.16864538.1693 537.1620 1.2 6.33 353675 hydroxy-E-cinnamoyl) Vaccinoside536.15299 536.1530 535.1457 0.0 6.35 6705 Avicularin 434.08491 434.0857433.0785 1.9 6.38 512642 Vaccinoside 536.15299 536.1545 535.1472 2.86.47 9321 Procyanidin A 576.12678 576.1265 575.1192 −0.5 6.47 11610Procyanidin tetramer 1152.25355 1152.2511 1151.2438 −2.1 6.47 33495Procyanidin trimer 864.19016 864.1892 863.1819 −1.1 6.48 113767Avicularin 434.08491 434.0859 433.0787 2.4 6.56 9167544-Hydroxyphenyl-gentioside 434.14243 434.1441 433.1368 3.8 6.56 75593′,4′,4′″,5′,7,7″-Hexahydroxy-8,3″′- 542.12130 542.1229 541.1156 3.06.59 7805 biflavanone 3,5-Bis(3,4- 516.12678 516.1259 515.1186 −1.7 6.616367 dihydroxycinnamoyl)quinic acide Avicularin 434.08491 434.0859433.0786 2.2 6.78 1907961 2,4,6-Trihydroxyphenylacetic acid; 320.05322320.0541 319.0468 2.6 7.08 8233 2-O-(3,4-Dihydroxybenzoyl) DunalianosideB 450.11621 450.1165 449.1092 0.6 7.42 19468 Procyanidin A 576.12678576.1246 575.1173 3.9 7.49 6252 Lyonside 552.22068 552.2212 551.2139 0.97.50 42922 Quercetin 3-glycosides; 592.14282 592.1432 591.1359 0.7 7.7315267 Monosaccharides, 3-O-[3-Hydroxy- 3-methylglutaroyl-(4)-I ± L-rhamnopyranoside] Leptosin 462.11621 462.1171 461.1098 1.9 8.39 50978-[5-(3,4-Dihydroxy-7-hydroxy-4- 510.13147 510.1324 509.1251 1.8 8.638411 oxo-2H-1-benzopyran-2-yl)-2- hydroxyphenyl]-2,3-dihydro-7-hydroxy-2-(4-hydroxyphenyl)-4H-1- benzopyran-4-one Lyoniside 552.22068552.2210 551.2138 0.6 8.73 6492 Procyanidin A 576.12678 576.1270575.1197 0.4 8.86 8440 Procyanidin B 578.14243 578.1420 577.1347 −0.812.84 7997

Multiple procyanidins were found in E2 at substantially higher contentcompared to E1. Procyanidin dimers—including both A and B types—werefound to be about fifty (50) times higher in E2 compared to E1 based ondetector counts with mass-to-charge ratio (‘m/z’) at 575.11 and 577.13.Procyanidin trimers with observed m/z at 863.18 were present at abouttwenty-three (23) times higher in E2 compared to E1, whereas procyanidintetramer with m/z at 1152.24 was over seven hundred (700) times higherin E2 compared to E1.

Similar bioflavonoids were also identified in E2 with much higherabundance, including isoquercetin, quercetin-3-arabinofuranoside,kaempferol glycoside, etc. based on LCMS analysis. Flavonoids withobserved m/z at 463.093—identified with molecular formula C₂₁H₂₂O₁₀— aretwenty (20) times higher for peak with retention time (‘RT’) at 6.38min, and thirty-six (36) times higher for peak with RT at 6.78 min forE2 compared to corresponding peaks detected in E1. Overall detectorcounts of flavonoids in E2 are over twenty (20) times higher thanflavonoids in E1 based on LCMS analysis.

LCMS TIC, PDA 280 nm, and PDA 350 nm chromatograms are provided in FIG.3 for E1 and FIG. 4 for E2. LCMS TIC chromatograms comparison between E2and E1—illustrated in FIG. 5—clearly showed the higher contents forprocyanidins and bioflavonoids in E2, while higher organic acid contentwas seen in E1 (FIG. 3).

Example 3—Anthocyanins Quantification

Anthocyanins quantification method was adapted from published HPLCanalytical method (J. AGRIC. FOOD CHEM., “Separation, identification,quantification, and method validation of anthocyanins in botanicalsupplement raw materials by HPLC and HPLC-MS”, Vol. 49(8), pp. 3515-3521(2001)). HPLC system used was an Hitachi D7000 HPLC system, with aPhenomenex Luna 10 μm C18 column having a column size of 4.6×250 mm.Solvents used in the mobile phase were 0.5% phosphoric acid in H₂O(Solvent A) and H₂O/ACN/Acetic Acid/H₃PO₄ (50%:48.5%: 1.0%:0.5%)(Solvent B). UV wavelength was 480 nm.

Reference standard cyanidin-3-glucoside was purchased from ChromaDex(Chicago, Ill. US). Cyanidin-3glucoside was prepared at 1 mg/mLconcentration in 2% (v/v) HCl in methanol solution in 5 mL volumetricflask. The stock solution was further diluted by ⅕, 1/10, 1/20, and1/100 times in 2% (v/v) HCl in methanol to give cyanidin-3-glucosidesolutions at five concentrations of 1.00, 0.20, 0.10, 0.05 and 0.01mg/mL, respectively. The five solutions were unitized to generate acalibration curve. Each sample was injected at 10 μL in threereplicates. The calibration curve was determined based on the integratedpeak areas. The correlation coefficient (R2) value ofcyanidin-3-glucoside was determined at 0.9985.

Samples were prepared for analysis as follows, 12.5, 25.0, 50.0, and100.0 mg of E1 were weighed. 1 mL of 2% (v/v) HCl in methanol was addedto each sample, and then each sample was mixed by sonication for fifteen(15) minutes and vortexed at 10,000 rpm for five (5) minutes. 20 μL ofsupernatant of each solution was injected to HPLC in three replicates.Quantitative analysis of five (5) anthocyanin compounds at differentconcentrations demonstrated linearity with correlation coefficients R²from 0.9953 to 0.9982 (FIG. 7). The amount of each individualanthocyanin was calculated based on the integrated peak areas againstcyaniding-3-glucoside at 0.05 mg/mL for the samples at a concentrationof 25 mg/mL and 50 mg/mL, respectively.

Five anthocyanins were quantified in E1 with a total content of 1.903mg/g as of dry weight of E1. These anthocyanins includedCyanidin-3-galactoside (‘C3Gla’), Cyanidin-3-arabinoside (‘C3-Ara’),Peonidin-3-galactoside (‘P3-Gla’), Peonidin-3-arabinoside (‘P3-Ara’),and Malvidin-3-galactoside (‘Mal3-Gla’), based on analysis andcomparison with those disclosed in the analytical method article and thearticle J. AOAC INT., “Determination of anthocyanins in Cranberry fruitand Cranberry fruit products by High-Performance Liquid Chromatographywith Ultraviolet Detection; Single-Laboratory Validation”, Vol. 94(2);pp. 459-466 (2011). These compounds are illustrated in FIG. 6. Noanthocyanins were detected in E2.

TABLE 3 Amount of five anthocyanins calculated in E1 R1 - 25 R1 - 50mg/g mg/mL mg/mL C3-Gla 0.506 0.503 C3-Ara 0.276 0.275 P3-Gla 0.5910.587 P3-Ara 0.200 0.195 Mal-3-Gla 0.330 0.331

Examples—Bioassay

Extracts of Cranberry fruit and Cranberry leaf were prepared withfood-grade ethanol, and then filtered and dried as described above.Research grade reagents were used for the rest of the assaypreparations. Extracts were dissolved in dimethyl sulfoxide (‘DMSO’) toa final concentration of 50 mg/mL, and then diluted in appropriatebuffer for each bioassay to working concentrations.

Example 4—DNA Damage Assay

When DNA damage—whether endogenous or exogenous—forms double strandedbreaks (‘DSBs’), it is always followed by phosphorylation of the histoneH2AX. H2AX is a variant of the H2A protein family, which is a componentof the histone octomer in nucleosomes. It is phosphorylated by kinasessuch as ataxia telangiectasia mutated (‘ATM’) and ATM-Rad3-related(‘ATR’) in the PI3K pathway. The protein γ-H2AX is the first step inrecruiting and localizing DNA repair proteins. DSBs can be induced bymechanisms such as ionizing radiation or cytotoxic agents andsubsequently, γ-H2AX foci quickly form. These foci represent the DSBs ina 1:1 manner and can be used as a biomarker for damage. An antibody canbe raised against γ-H2AX, which can therefore be visualized byimmunofluorescence through secondary antibodies The detection andvisualization of γ-H2AX by flow cytometry allow the assessment of DNAdamage, related DNA damage proteins, and DNA repair.

Human skin fibroblasts were seeded at a density of 8000 cells/well in96-well tissue culture plates. After 24 hours, cells were treated withtest compounds for 48 hours, after which the cells were treated with 1mM hydrogen peroxide for 4 hours to induce DNA damage. Hydrogen peroxideinduces DNA damage by creating breaks in the DNA. Cells were then fixedand stained with an antibody to the biomarker γ-H2AX, which is aphosphorylated histone variant that is found at double-strand breaks. Atsites of double-strand breaks, the histones are phosphorylated,indicating that the DNA is damaged and requires repair. An antibody forγ-H2AX—the phosphorylated histone variant—is useful in identifying sitesof DNA damage. Nuclei were stained with 4′,6-diamidino-2-phenylindole(‘DAPI’), which is a fluorescent stain that binds to DNA. Pictures weretaken with Image Xpress and analyzed with Meta Xpress to measurefluorescence intensity in each condition divided by the total number ofcells.

DNA damage in response to hydrogen peroxide treatment was measured bythe amount of γ-H2AX present in cells. Cranberry fruit E1 and Cranberryleaf E2 extracts were tested for DNA damage inhibition at 50 and 100μg/mL based on the amount of γ-H2AX detected after treatment withhydrogen peroxide to induce DNA damage. Catechin and soliprin at 25μg/mL were used as positive controls. As shown in FIG. 10, cranberryleaf extract E2 at 100 μg/mL exhibited significant inhibition of DNAdamage. Percent inhibition was calculated relative to wells that werenot treated with extracts but were exposed to hydrogen peroxide. Fromthis Example, it is seen that pre-treating fibroblasts with E2 at 100μg/mL significantly reduced the amount of γ-H2AX detected, showing theability of E2 to protect DNA from damage.

Cell Counting Kit-8 (‘CCK-8’) was used to determine the percentage ofviable cells in each treatment relative to an untreated control. CCK-8reagent was added to the media (final concentration 10% total volume)and incubated for one (1) hour at 37° C. and 5% CO₂ before absorbancewas read on a Multimode Reader at 460 nm to determine the number ofviable cells relative to untreated control wells. None of the treatmentswas statistically significantly different from the untreated controls(FIG. 11).

The above data illustrates that the botanical extract of the leaf ofVaccinium macrocarpon has one or more compounds that exhibitanti-oxidant activity. More particularly, the cranberry leaf extract mayhave reasonable activities in ameliorating γ-H2AX activity.

The above description discloses several methods and materials of thepresent invention. This invention is susceptible to modifications in themethods and materials, as well as alterations in the fabrication methodsand equipment. Such modifications will become apparent to those skilledin the art from a consideration of this disclosure or practice of theinvention disclosed herein. Further, unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs. Consequently, it is not intended that this inventionbe limited to the specific embodiments disclosed herein, but that itcover all modifications and alternatives coming within the true scopeand spirit of the invention as embodied in the attached claims.

We claim:
 1. A composition comprising the botanical extract of the leafof Vaccinium macrocarpon, wherein the botanical extract inhibits γ-H2AXactivity.
 2. The composition according to claim 1, wherein the botanicalextract is present in an amount of about 1.0 μg/mL or greater.
 3. Thecomposition according to claim 2, wherein the botanical extract ispresent in an amount of about 1.0 μg/mL to about 1000.0 μg/mL.
 4. Adietary supplement having antioxidant properties comprising a cranberryleaf extract in a therapeutically effective amount, wherein thecranberry leaf extract inhibits γ-H2AX activity.
 5. The dietarysupplement according to claim 4, wherein the cranberry leaf extract ispresent in an amount of about 25.0 μg/mL, to about 500.0 μg/mL.
 6. Amethod of inhibiting oxidation-induced DNA damage in a subjectcomprising administering a composition comprising the botanical extractof the leaf of Vaccinium macrocarpon at a concentration of about 1.0μg/mL to about 1000.0 μg/mL.